Loss of WHY1 has Specific Effects on Leaf Transcript Abundance
Knockdown of the WHIRLY protein clearly delayed greening of emerging
barley leaves (Fig. S2). To determine whether the WHIRLY protein
influences other aspects of leaf development, a comparative
transcriptome analysis of basal, mid and tip sections of leaves of wild
type and two whirly knockdown lines was conducted. Significant
differences in transcript abundance (P<0.05) based upon leaf
position, genotype or the interaction of the two factors was determined
using 2-way analysis of variance which revealed 1732 transcripts
dependent on line, 2240 transcripts dependent on leaf region and only 23
which exhibited a distribution in abundance based on an interaction
between the two factors (Table S2).
Of the 23 transcripts exhibiting a genotype by leaf region interaction
in their patterns of abundance, three transcripts (AK370975,
MLOC_56051.1, MLOC_56052.1) that exhibited homology to chlorophyll
binding proteins had a low abundance in all sections of wild type leaves
and although at higher abundance in W1-1 leaves both genotypes exhibited
a pattern of reducing abundance from base to tip. In contrast, W1-7
leaves exhibited a reverse pattern of abundance increasing from base to
tip. A similar pattern of abundance was observed for a transcript
encoding a thylakoid luminal protein (AK370198). These data are
consistent with delayed assembly of the photosystems in WHY-deficient
leaves. Two other transcripts encoding proteins with functions in
plastid biogenesis and metabolism, respectively also exhibited a
genotype by leaf region dependent expression pattern. MLOC_9203.2
encodes a tubulin-like protein with homology to Arabidopsis AT2G36250
encoding an FtsZ protein essential for chloroplast division (Osteryoung,
Stokes, Rutherford, Percival, & Lee, 1998) while MLOC_69205.1 encodes
an oxaloacetate/malate antiporter acting as a malate valve to balance
NADPH/ATP ratios in the plastid (Selinski, & Schiebe, 2019).
We then compared the transcriptome profiles of the three developmental
regions in the W1-1 and W1-7 leaves with those of the wild type leaves
focusing on the key transcripts discussed above in the wild type
developmental pattern (Fig. 4, Table S3). Of these transcripts, most
exhibited similar patterns of abundance in the developmental profiles of
all genotypes. Notable exceptions were MLOC_70809.1 encoding a
homologue of Arabidopsis GATA, NITRATE-INDUCIBLE, CARBON METABOLISM
INVOLVED (GNC) transcription factor (AT5G56860) that regulates stomatal
development, greening and chloroplast development, MLOC_53744.1
encoding NAC1, a senescence associated transcription factor under the
control of auxin, SAG12 (MLOC_47161.1) and two transcripts encoding ARF
transcriptions factors (AK364144, MLOC_73144.4) with functions in leaf
morphogenesis and development. The patterns of abundance of these
transcripts were perturbed in the absence of WHIRLY1 relative to wild
type. While SAG12 transcripts were more abundant in all the sections of
the W1-7 line than the other genotypes, the ARF and NAC1 transcripts
were less abundant in all sections relative to the other genotypes (Fig.
4). These differences are indicative of divergent developmental
programmes in leaves deficient in the WHY1 protein.
As a key phenotype of WHY1 knockdown was reduced and delayed greening
(Fig. S2) we next sought to examine the shift in abundance of
transcripts associated with light signalling and plastid development in
leaf regions of different age. Twenty-nine transcripts associated with
light dependent plastid biogenesis were identified in the developmental
profile of the wild type, of which 22 exhibited a high to low gradient
of abundance from the leaf base to leaf tip (Fig. 5). Six transcripts
encoded chloroplastic ribosomal proteins and a further six had putative
functions in plastid gene expression. Three transcripts (MLOC53063.1,
AK3635024 and AK363292) exhibited similarity to AT2G03200 which encodes
a putative chloroplast nucleoid DNA binding protein while a further two
transcripts (MLOC_65040.1 and MLOC_69013.1) exhibited similarity to
AT5G10770 containing similar domain structures (Table S4). The plastid
nucleoids comprise multiple copies of DNA, RNA and a range of proteins
with functions in replication, gene expression and DNA binding that are
believed to control plastid gene expression in a way analogous to
chromatin (Powikrowska, Oetke, Jensen, & Krupinska, 2014). A further
transcript MLOC_9558.1 encoded a protein with similarity to plastid
encoded RNA polymerase alpha subunit, essential for light dependent
plastid biogenesis (Yoo et al., 2019). The levels of this transcript
declined from base to tip in wild type and WHY 1-1 but the levels of
this transcript were high in the base and tip regions of the WHY1-7
leaves.